The effects if the intra- and inter-molecular interactions betweenthe domains of the HCV NS3 and NS4A on the RNA helicase activity

• Main Authors: Wan-Fen Kuang, 鄺菀芬
• Other Authors: Lih-Hwa Hwang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/80143608033464917845

碩士 === 國立臺灣大學 === 微生物學研究所 === 91 === The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) possesses three activities which are considered essential for virus replication: a serine protease locating at the N terminus and helicase and NTPase activities locating at the C terminus. Thus, it emerges as an attractive target for drug design. By far, most studies investigated the protease activities or RNA helicase activities by using separated domains which, though were functional, did not represent true biological entity. In order to investigate the effects of the intra-molecular interactions between the two domains of NS3 and the inter-molecular interactions between NS3 and NS4A on the RNA helicase activity, we produced truncated NS3 that contained only the helicase domain (NS3H), full-length NS3, NS3-NS4A and its mutants, using a Baculovirus expression system. These proteins were used for the analysis of helicase, ATPase, and protease activities. Our data showed that the N terminal protease domain of the HCV NS3 inhibited the RNA helicase activity. However, the presence of NS4A in the NS3-NS4A complex reversed the inhibitory effects. The reason why NS3 displayed lower helicase activity than NS3H or NS3-NS4A was not due to lower ATPase activity or RNA binding activity, but due to lower processivity of RNA unwinding. The optimal range of pH or divalent cation concentration for all three enzymes appeared to be similar. However, NS3H exhibited higher sensitivity to monovalent cation than NS3 or NS3-NS4A, both of which could stand up to 100 mM. Mutant K1236N, which had a mutation at the conserved helicase motif I, lost the ATPase activity and thus the helicase activity, but its protease activity was not affected. Mutant R1487Q, which had a mutation in the arginine-rich motif, had similar ATPase and RNA binding activities as NS3-NS4A, but its helicase and protease activities were reduced. In the second part of this study, the helicase activities of 1193, a truncated protein that only contained the NS3 helicase domain, and its five mutants were tested. According to the comparison between the structures of 1193 and PcrA helicase, amiano acids Q1606、K1609、R1613、K1615、Q1632 could be the residues that interact with dsRNA. Therefore, we generated five muatants that had single point mutation at each site, and tested the helicase activities of these mutant proteins. The preliminary results showed that mutant K1632E might have reduced helicase activity.